The proposed research is a continued study of the involvement of proteins in splicing group I and II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins to help fold the intron RNA into the catalyti- cally active structure. Previously, we found that a key protein required for splicing group I introris in Neurospora crassa mitochondria is the mitochondrial (mt) tyrosyl-tRNA synthetase (TyrRS;CYT-18 protein). Structural studies during the current grant period showed that group I introns bind to the TyrRS's nucleotide-binding fold on the side opposite that which binds tRNATyr and use a new RNA-binding surface formed by three separate insertions and other structural adaptations relative to non-splicing bacterial TyrRSs. Moreover, these structural adaptations appear uniquely characteristic of the mt TyrRSs of a family of fungi that includes important human and plant pathogens. In the proposed research, we would continue to study the mechanism of action of CYT-18, how it evolved to function in RNA splicing, and whether splicing-active fungal mt TyrRSs might be a target for antifungal drugs. For group II introns, we developed an experimental system based on the mobile Lactococcus lactis LI.LtrB intron, which en- codes a reverse transcriptase (RT) that functions both in intron mobility and as an intron-specific splicing factor ("maturase"). During the current grant period, we delineated interacting regions of the protein and intron RNA, which in conjunction with structural models, suggest specific hypotheses about how RT/maturases stabilize the active RNA structure and evolved to function in splicing. In the proposed research, we would use biochemical, genetic, and structural approaches to test these hypotheses and obtain the first comprehensive picture of how a group II in- tron RT functions in RNA splicing. In addition to proteins that stabilize the active RNA structure, we found that the efficient splicing of mt group I and II introns requires DEAD-box proteins and obtained evidence that they function as RNA chaperones to disrupt stable, inactive structures that are "kinetic traps" in RNA folding. These proteins, CYT-19 in N. crassa and Mss116p Saccharomyces cerevisiae, also function in other RNA processing reactions and in mt translation. Our findings suggest that CYT-19 and Mss116p may be the founding members of a class of DExH/D- box proteins that act broadly as RNA chaperones on structurally diverse RNAs and RNA/protein complexes, and they raise the possibility that DExH/D-box proteins that function similarly as general RNA chaperones exist and play an important role in RNA metabolism in all organisms. In the proposed research, we would test these hypotheses and use the facile group I and group II intron splicing assays to further study and define the structural and functional characteristics of DExH/D-box proteins that act as general RNA chaperones. Finally, we will continue a yeast genetic screen to identify novel group I and II intron splicing factors, particularly those for the group II introns a!5v and bll By combining these splicing factors with DEAD-box RNA chaperones, we hope to reconstitute the complete splicing apparatus for these important model group II introns. This research is intended to provide novel information about how proteins mediate RNA folding and RNA-catalyzed reactions, the evolution of introns and splicing mechanisms, and the function and evolution of aminoacyl-tRNA synthetases, reverse transcriptases, and DExH/D-box proteins, all relevant to human diseases.